Published online August 9, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 14 4217–4223
doi:10.1093/nar/gkh753
Importance in catalysis of a magnesium ion with
very low affinity for a hammerhead ribozyme
Atsushi Inoue1,3, Yasuomi Takagi1,2 and Kazunari Taira1,2,3,*
1
Gene Function Research Center and 2iGENE Therapeutics, Inc., National Institute of Advanced Industrial Science
and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan and 3Department of
Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
Received December 16, 2003; Revised and Accepted July 21, 2004
ABSTRACT
Available evidence suggests that Mg2+ ions are
involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether
divalent metal ions really function as catalysts
when they are present. We investigated ribozyme
activity in the presence of high levels of Mg2+ ions
and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly
with increasing concentrations of Mg2+ ions and did
not reach a plateau value even at 1 M Mg2+ ions.
Furthermore, this dependence on Mg2+ ions was
observed in the presence of a high concentration of
Li+ ions. These results indicate that the Mg2+ ion is
a very effective cofactor but that the affinity of the
ribozyme for a specific Mg2+ ion is very low.
Moreover, cleavage by the ribozyme in the presence
of both Li+ and Mg2+ ions was more effective than
expected, suggesting the existence of a new reaction
pathway—a cooperative pathway—in the presence of
these multiple ions, and the possibility that a Mg2+ ion
with weak affinity for the ribozyme is likely to function
in structural support and/or act as a catalyst.
INTRODUCTION
Naturally existing catalytic RNAs include hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS)
ribozymes, group I and II introns, and the RNA subunit of
RNase P (1–7). In addition, recent structural and chemical
analyses strongly suggest that ribosomal RNA might also
be a ribozyme (8–11). In addition, the possibility exists that
the RNA component of the spliceosome might be a ribozyme
too (12). The earliest research on ribozymes suggested that all
ribozymes might be metalloenzymes that require divalent metal
ions, in particular Mg2+ ions, for catalysis, and that all might
operate by a basically similar mechanism. However, subsequent, extensive studies revealed that the catalytic activity
of hairpin ribozymes is independent of divalent metal ions
(7,13–18). Thus, the various types of ribozymes appear to
exploit different cleavage mechanisms, which depend upon
the architecture of the individual ribozyme. Even hammerhead
ribozymes, which have generally been characterized as typical
metalloenzymes, can no longer be categorized unambiguously.
Naturally existing hammerhead ribozymes were originally
identified in some RNA viruses, and it was demonstrated that
they act in cis during viral replication by the rolling circle
mechanism (3). In the laboratory, ribozymes have been engineered such that they act in trans against other RNA molecules
and catalyze the cleavage of phosphodiester bonds at specific
sites to generate specific products, each of which has a 20 ,30 cyclic phosphate and a 50 -hydroxyl group (19–22). The transesterification mechanism includes deprotonation of the
20 -hydroxyl moiety of a ribose group, nucleophilic attack of
the 20 -oxygen on the adjacent phosphorus atom, and protonation of the 50 -oxyanion leaving group (Figure 1A). A large
body of evidence also indicates that the P9/G10.1 site binds a
metal ion with high affinity, with other metal ion-binding sites
being located around the G5 nucleobase and A13 phosphate
near the site of cleavage (23–36). Thus, the idea that ribozymes are metalloenzymes has been generally accepted. However, it was reported recently that hammerhead ribozymes are
active in the presence of very high concentrations of monovalent cations, such as Li+ or NHþ
4 ions, in the absence of
divalent metal ions (37). This finding raises the possibility
that hammerhead ribozymes should not be classified as metalloenzymes. Therefore, we decided to investigate ribozyme
activity in the presence of Mg2+ ions and the effects of Mg2+
ions in the presence of Li+ ions on ribozyme activity, using a
well-studied model hammerhead ribozyme (R32) and its
substrate (S11), both of which are shown in Figure 1B
(5–7,25,28,32–34,38–41).
We investigated the dependence on the concentration of
Mg2+ and Li+ ions of ribozyme activity over a range from
5 mM to 1 M Mg2+ ions and 1 to 5 M Li+ ions, respectively.
Although several research groups have reported similar analyses (42–50), the concentrations of Mg2+ ions used in most of
these studies were <100 mM, and little information is available
about activity at a higher concentration, such as 1 M. The
activity at high concentrations of Mg2+ ions, as compared
*To whom correspondence should be addressed at Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo
113-8656, Japan. Tel: +81 3 5841 8828 or +81 29 861 3015; Fax: +81 29 861 3019; Email: [email protected]
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Nucleic Acids Research, Vol. 32 No. 14 ª Oxford University Press 2004; all rights reserved
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Nucleic Acids Research, 2004, Vol. 32, No. 14
Figure 1. (A) Schematic representation of the proposed mechanism of the hammerhead ribozyme reaction. The 20 -hydroxyl moiety is activated by the catalyst and
attacks the adjacent phosphate nucleophilically, with subsequent cleavage of the bond at the 50 -oxygen. The developing negative charge on the leaving 50 -oxygen is
stabilized by another catalyst. (B) The sequences and secondary structures of the hammerhead ribozyme (R32) and substrate (S11) used in this study. (C) The
proposed two-stage folding scheme for the hammerhead ribozyme–substrate complex. The higher-affinity Mg2+ ion(s) drives the formation of domain II, which
contains non-Watson–Crick pairings and the lower-affinity Mg2+ ion(s) rotates around helix I, forming the catalytic core.
to physiological concentrations, is useful for studies of the
chemistry of the hammerhead ribozyme and it allowed us
to estimate the intrinsic cleavage rate constant and to
compare an enzymatic reaction to a non-enzymatic reaction.
We were also able to calculate acceleration energy more
precisely and to investigate the dependence on Mg2+ ions
in the presence of high concentrations of Li+ ions. Considering
the results of such analyses, we propose the existence of a new
cooperative pathway that involves divalent metal ions, such
as Mg2+, and monovalent ions, such as Li+, in the reaction
catalyzed by a hammerhead ribozyme. We also discuss the
nature of the catalysts.
MATERIALS AND METHODS
Preparation of the hammerhead ribozyme and its
substrate
The ribozyme (R32) and its substrate (S11) were synthesized
chemically on a DNA/RNA synthesizer (model 394; PE
Applied Biosystems, Foster City, CA) using phosphoramidic
chemistry with 20 -tert butyldimethylsilyl (TBDMS) protection
as described previously (25). Chemically synthesized oligonucleotides were deprotected in a mixture of 28% ammonia
and ethanol (3:1) at 55 C for 8 h. The mixture was evaporated
to dryness and the residue was allowed to dissolve in 1 ml of
1 M tetrabutylammonium fluoride (TBAF; Sigma-Aldrich,
Japan K.K., Tokyo, Japan) at room temperature for 12 h.
After the addition of 1 ml of water, the mixture was desalted
on a gel-filtration column (Bio-Gel P-4; Bio-Rad Laboratories,
Hercules, CA). Fully deprotected oligonucleotides were purified by gel electrophoresis on a 20% polyacrylamide gel that
contained 7 M urea, the respective bands were excised from
the gel, and oligonucleotides were extracted in water. The
oligonucleotides were recovered by ethanol precipitation
and then solutions were desalted on a gel-filtration column
(TSK-GEL G3000PW; TOSOH, Tokyo, Japan) by highperformance liquid chromatography (HPLC) with ultrapure
water. All of the RNA oligomers were quantitated in terms
of absorbance at 260 nm.
The substrate S11 was labeled with [g-32P]ATP by T4 polynucleotide kinase (Takara Bio, Inc., Shiga, Japan). Radiolabeled S11 was purified on a 20% polyacrylamide gel that
contained 7 M urea and then purified by the standard procedure, as described above, with desalting on a gel-filtration
column (NAPTM-10 column; Amersham Biosciences, K.K.,
Tokyo, Japan).
Quantification of the ribozyme reaction
All ribozyme reactions were performed under single-turnover
conditions to ensure that conversion of the ribozyme–substrate
Nucleic Acids Research, 2004, Vol. 32, No. 14
complex to the ribozyme–product complex could be monitored kinetically without complications due to complex formation and slow release of products. The solution for the
ribozyme reaction contained a trace amount of 50 -32P-labeled
S11 and 25 mM Bis-Tris buffer at pH 6.0 and 25 C. The pH
values of all 1.25· pre-stock Bis-Tris buffers that contained
appropriate metal ions (metal-ion–buffer) were adjusted
appropriately with HCl and we confirmed that each buffer
had the appropriate pH under the chosen reaction conditions.
Each reaction was initiated by addition of the substrate to a
mixture of metal-ion–buffer and ribozyme, and aliquots were
removed from the reaction mixture at appropriate intervals.
Each aliquot was mixed with more than three volumes of a
stop solution that contained 100 mM MES (pH 6), 100 mM
EDTA, 7 M urea, xylene cyanol (0.1%) and bromophenol blue
(0.1%), and then it was stored at 80 C prior to analysis. Since
EDTA does not chelate Mg2+ and Li+ ions efficiently at lower
pH values, we confirmed that reactions did not continue in the
stop solution and that quenching was effective due to high
concentration of urea in this solution. Uncleaved substrate and
50 -cleaved products were separated on a 20% polyacrylamide
gel that contained 7 M urea. The extent of each cleavage
reaction was quantitated with an imageanalyzer (Storm 830;
Molecular Dynamics, Sunnyvale, CA). For each reaction, an
observed rate constant was determined by non-linear leastsquares fitting of the time course of reaction using a
pseudo-first-order equation.
4219
A
B
RESULTS AND DISCUSSION
The dependence of the activity of the hammerhead
ribozyme on the concentration of Li+ ions
We examined the dependence of the activity of the ribozyme
on the concentration of Li+ ions to confirm that Li+ ions affect
the ribozyme’s activity, as reported previously by others (51).
We performed reactions under single-turnover conditions at
pH 7.5 and 25 C. The results are shown in Figure 2A.
Although the activity reached a plateau at 3 M Li+ ions,
the dependence on the concentration of Li+ ions was observed
(with a slope of three) below the plateau. In the study by
O’Rear et al. (51), the hammerhead ribozyme reaction exhibited second-order dependence on Li+ ions up to 4 M at pH 7.5.
Although our curve is steeper than the curve that they obtained
under the same conditions, with the exception of the concentrations of Li+ ions, the slopes of both profiles obtained with
Li+ ions are clearly steeper than those of profiles obtained with
Mg2+ ions. Our profile is unique insofar as we observed saturation of the ribozyme reaction in the range of 3–5 M
(Figure 2A). This plateau suggests that the ribozyme reaction
might involve the binding of Li+ ions to the ribozyme–
substrate complex.
We also confirmed the dependence on pH, with a slope of
unity, of the activity in the presence of a high concentration of
Li+ ions, as shown in Figure 2B. This result supports the
hypothesis that the cleavage step is the rate-limiting step
even at such a high ionic strength. The pH profile is also
consistent with the previous report by Curtis and Bartel
(52). As shown in Figure 2A, the observed rate constant at
a saturating concentration of Li+ ions (3–5 M) at pH 7.5 was
0.17 min1. The estimated observed rate constant in the
Figure 2. (A) Dependence of cleavage activity on the concentration of Li+ ions.
The activity increased linearly with the concentration of Li+ ions until 3 M.
Reaction conditions: 25 mM Bis-Tris, pH 7.5, and varying concentrations of Li+
ions as indicated. (B) Dependence on pH of the activity in 2 M Li+ ions. The Li+containing buffer used in this experiment included Bis-Tris propane and Tris.
The cleavage rate increased linearly with pH, yielding a slope of 0.9.
presence of 800 mM Mg2+ ions was 32 min1 at pH 7.5, as
determined from the value of 1 min1 at pH 6 at the same
temperature as that at which the Li+ experiment was performed
(see later). The difference was, thus, approximately 200-fold
under similar conditions. The true difference might be even
greater because, in contrast to the results with Li+, the rate at
800 mM Mg2+ does not reach a plateau value (see later). These
data indicate clearly that Mg2+ ions are more suitable for an
effective hammerhead ribozyme reaction than any other
monovalent ions (Li+ ions have the highest activity of all
monovalent ions in the ribozyme reaction).
The dependence of the activity of the hammerhead
ribozyme on the concentration of Mg2+ ions
We attempted first to determine how many Mg2+ ions might be
involved in our model ribozyme reaction and the saturating
concentration of Mg2+ ions in the reaction, beyond which the
cleavage rate constant no longer increases. We examined the
dependence on the concentration of Mg2+ ions of the activity
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Nucleic Acids Research, 2004, Vol. 32, No. 14
Figure 3. The Mg2+-titration curve for cleavage activity in the presence (open
circles) and absence (filled circles) of Li+ ions. In the absence of Li+ ions, the
line drawn with the linear best fit had a slope of 0.7. The activity increased with
increasing concentrations of Mg2+ ions and did not reach a plateau value even at
800 mM Mg2+ ions. In the presence of 2 M Li+ ions, the activity increased with
increases in the concentration of Mg2+ ions up to 600 mM. Triangles are the
calculated rates in the presence of Mg2+ ions plus 2 M Li+ ions, which were
determined simply by adding the respective reaction rates together. Since the
observed rate in the presence of 2 M Li+ ions only at pH 6 was <0.01 min1,
triangles are at almost the same positions as the filled circles. The rates observed
in the presence of Mg2+ ions and Li+ ions together (open circles) were clearly
higher than the calculated values (triangles).
of the R32 hammerhead ribozyme up to 1 M Mg2+ ions. We
performed reactions with R32-S11 (Figure 1B) under singleturnover conditions with a saturating amount of ribozyme with
respect to the amount of S11 for the same reasons as noted
above. The reaction in the presence of Mg2+ ions is accelerated
with increases in pH, with a slope of unity (7,40,43,44,47,53).
We adjusted the pH of reactions to 6.0 to slow down the
reaction so that we could determine the rate constants of
rapid reactions precisely. As shown by closed circles in
Figure 3, the dependence on Mg2+ ions was approximately
first-order. However, no plateau was reached under our conditions, even above 800 mM Mg2+ ions. The continuous
increase in rate constant upon the addition of more and
more Mg2+ ions indicates the involvement of one Mg2+ ion
that has low affinity for the hammerhead ribozyme–substrate
complex. At 800 mM Mg2+ ions, the rate constant approached
1.1 min1 at pH 6.0, and this is the limit of detection of a rapid
cleavage reaction under standard laboratory conditions.
Analyses of the structure of hammerhead ribozymes and of
the conformational changes caused by interactions with Mg2+
ions have indicated that two major conformational changes
occur: the formation of domain II, which is followed by
the formation of domain I, as shown in Figure 1C (38,45,
48,50,54). The formation of domain II results in coaxial stacking of helices II and III, induced by the binding of a higheraffinity Mg2+ ion(s) to P9 phosphate and N7 of G10.1 (P9/
G10.1) of the ribozyme–substrate complex (38,45,48,50,54–
58). The second transition is the formation of the catalytic
domain with movement of stem I toward stem II, which is
induced by the binding of a lower-affinity Mg2+ ion(s). The Kd
values of the two domains have been determined by various
methods to be several hundred micromolar and several millimolar, respectively (45,46,48,50,54). Thus, at several hundred
millimolar Mg2+ ions, the formation of domains II and I should
be complete.
Taking this structural information into consideration, we
can reasonably conclude that the very-low-affinity Mg2+ ion
that we detected in the present study might be involved in
some step other than the formation of domains I and II. This
step might be a conformational change or the binding of a
catalytic species to the ribozyme–substrate complex. Walter
and coworkers recently reported that a third and previously
undetected metal ion at rather high concentrations might play a
role in the induction of a minor conformational adjustment that
leads to the formation of the active state after the formation of
domains I and II (59). The Mg2+ ion that we detected had very
low affinity, and the relative level of truly active ribozyme
species at a concentration of several millimolar Mg2+ ions
corresponded to <1% of all the ribozyme–substrate complexes
in the reaction mixture [compare 1 min1 in 10 mM MgCl2 at
pH 8 and 25 C (60) with 100 min1 in 800 mM MgCl2 at the
same pH and the same temperature (see later)].
One might agree that Figure 3 does not support the existence
of a very-low-affinity binding site for an Mg2+ ion on the
ribozyme–substrate complex since the binding curve does
not reach a plateau. If the dependence that we observed
here were due only to ionic strength, no plateau would be
observed. However, we did observe a plateau in the case of
Li+ ions (Figure 2A). Because reactions were so rapid, even at
low pH, we could not monitor the kinetics at even higher
concentrations of Mg2+ ions, such as 3 M. However, it is likely
that, in ribozyme reactions, ionic strength is less important
than the ion radius and/or the electron density of metal ions
since (i) a correlation between the ion radius of Group I monovalent metal ions and ribozyme activity has been observed
(52,57) and (ii) an apparent plateau was reached in the presence of high concentrations of Mg2+ ions plus 2 M Li+ ions
(Figure 3). Although we cannot calculate a Hill constant for
Mg2+ ions and estimate the number of binding sites for Mg2+
ions from our current data, all the available data strongly
support the possibility of the existence of a very-low-affinity
metal-binding site(s).
Misra and Draper (61) proposed a model for the stabilization of RNA by Mg2+ ions that arises from two distinct binding
modes, diffuse binding and site binding. Diffusely bound Mg2+
ions are defined as fully solvated ions that interact with RNA
only through long-range electrostatic interactions. Site-bound
Mg2+ ions are defined as partially desolvated ions that are
attracted to electronegative pockets. In general, the affinity
of diffusely bound Mg2+ ions appears to be lower than that
of site-bound Mg2+ ions. Thus, it is possible that the very-lowaffinity Mg2+ ions that we detected here might be involved in
diffuse binding. Although diffuse binding can sometimes play
a dominant role in stabilizing the tertiary structures of small
RNAs (61), we cannot exclude the possibility that such diffuse
binding Mg2+ ion might play a role in catalysis in the ribozyme
reaction at a specific site in the ribozyme–substrate complex.
In 800 mM Mg2+ ions, the observed rate constant of the
reaction catalyzed by the hammerhead ribozyme can be
estimated to be 100 min1 at pH 8 and 25 C, from the
Nucleic Acids Research, 2004, Vol. 32, No. 14
dependence on pH with a slope of unity. The estimated rate
constant at concentrations of Mg2+ ions >800 mM should be
much higher than that at 800 mM since the cleavage reaction
did not reach saturation at 800 mM Mg2+ and approached the
estimated observed rate constant of a ‘kissing ribozyme’,
under saturating conditions with respect to Mg2+ ions and
pH (62).
The dependence of the activity of the hammerhead
ribozyme on Mg2+ ions in the presence of a high
concentration of Li+ ions
In order to investigate the properties of Mg2+ ions in the
ribozyme reaction in further detail, we examined the dependence on Mg2+ ions in the presence of a high concentration
(2 M) of Li+ ions. We chose the Li+ ion as the monovalent
cation because this ion is the most active of a large variety of
monovalent cations (57). At 2 M Li+ ions at pH 6.0, the
hammerhead ribozyme had detectable activity in the absence
of Mg2+ ions. We varied the concentration of Mg2+ ions from
5 to 900 mM and measured the rate constant. As shown by
open circles in Figure 3, we observed approximately firstorder dependence on the concentration of Mg2+ ions up to
600 mM, and then the rate constant started to reach a plateau
from 600 to 900 mM Mg2+ ions. These data indicate that, even
in the presence of a very high concentration of Li+ ions, an
Mg2+ ion with very low affinity plays an important role in the
hammerhead ribozyme reaction. The plateau that seemed to
appear in the presence of high concentrations of Li+ ions
(Figure 3, open circles) was not observed in the absence of
Li+ ions (closed circles). This apparent plateau was not due to
misfolding of RNA at such a high ionic concentration because
the extent of cleavage at the end of the reaction was always
>90% under these conditions. The plateau might indicate that
Li+ ions help the ribozyme–substrate complex to fold into a
more active form, contributing to the enhanced binding of the
very-low-affinity Mg2+ ion(s) to the complex.
We examined reactions in the presence of Mg2+ ions only
(Figure 3, closed circles) and in the presence of Li+ ions only
(57). We then calculated rate constants (Figure 3, triangles) on
the assumption that Mg2+ and Li+ ions function independently.
It should be noted that the observed rate constant in the presence of both Mg2+ ions and 2 M Li+ ions together (Figure 3,
open circles) was definitely higher than the calculated rates
(Figure 3, triangles) at pH 6 (compare open circles with
triangles in Figure 3). One might expect that, in such an
experiment (i) the activities in the presence of both metal
ions together would be lower than the estimated activities
(triangles) because of the lower ability of Mg2+ ions to bind
to RNA in the presence of high ionic strength due to 2 M Li+
ions and (ii) the stimulation by Mg2+ ions in the presence of
such a high concentration of Li+ ions would be smaller than in
the absence of Li+ ions. It has been reported that stimulation by
Cd2+ ions is reduced in a ribozyme reaction in the presence of
4 M Li+ ions (51), while stimulation by Cd2+ ions is observed
in reaction mixtures that contain 10 mM Ca2+ ions instead of 2
M Li+ ions (23). However, the results in the present study were
quite opposite: (i) the activities in the presence of Mg2+ ions
and 2 M Li+ ions together (open circles in Figure 3) were
higher than the calculated activities (triangles in Figure 3),
and (ii) the stimulation by Mg2+ ions in the presence of 2 M Li+
4221
ions (open circles in Figure 3) was even greater than that
by Mg2+ ions in the absence of Li+ ions (closed circles in
Figure 3).
Our observations suggest the existence of a new cooperative
pathway that involves Li+ and Mg2+ ions, in which both metal
ions function cooperatively in structural support and/or as the
catalyst(s) to increase the rate constant of cleavage (57). In
the case of cleavage of RNA by another type of ribozyme, the
RNA subunit of RNase P, a similar cooperativity between
the metal ions, e.g. Mg2+ and Ca2+, has been reported (63).
What is the catalyst in the hammerhead reaction?
To the best of our knowledge, this report is the first to describe
the dependence of ribozyme activity on a high concentration
of Mg2+ ions under single-turnover conditions (Figure 3). The
reaction did not reach a plateau even at 800 mM Mg2+ ions.
Under these conditions at pH 6, the observed rate constant was
1.1 min1. The activity of the ribozyme is known to depend on
pH, with a slope of unity. Thus, we can estimate an observed
rate constant for the ribozyme reaction of 110 min1 at pH 8.
It is very unlikely that this rate constant of cleavage can
occur without an effective catalyst(s) (64). So, what is the
catalyst(s) of the reaction?
In studies of solvent isotope effects on the ribozyme reaction in the presence of deuterium and Li+, Mg2+ or NHþ
4 ions,
we failed previously to observe any proton transfers in the
transition state during the ribozyme reaction in the presence
of either Li+ or Mg2+ ions but we did observe proton transfer in
the presence of NHþ
4 ions (53,65). We interpreted such results
by suggesting that metal ions, such as Mg2+ and Li+, function as
a Lewis acid catalyst while NHþ
4 ions function as a general acid
catalyst during the ribozyme reaction. Thus, the catalyst can
change according to the conditions around the ribozyme. On the
basis of this hypothesis and the involvement of two other kinds
of Mg2+ ions in the formation of domains I and II, it is possible
that the newly identified Mg2+ ion with very low affinity that we
observed in this study might be the true catalyst. Our novel
cooperative pathway might involve an Mg2+ ion in a catalytic
role after monovalent cations and other Mg2+ ions have acted
to generate the pre-active conformation just before chemical
cleavage by the hammerhead ribozyme. In the presence of
monovalent ions exclusively, monovalent ions can also function as the catalyst but they are not as effective. Mg2+ ions have a
higher charge density than Li+ ions and would function better as
catalyst in the reaction. Thus, when both Mg2+ and Li+ ions are
present in the reaction mixture, the high activity of the ribozyme
reaction is likely due to an Mg2+ ion catalyst. The Li+ ions act to
support the formation of the domains I and II in cooperation with
Mg2+ ions, as discussed above.
We are at present constructing a reaction scheme for ribozyme reactions in the presence of both Mg2+ and Li+ ions,
namely, a ‘cooperative pathway’, by determining the details of
the stoichiometric relationships among these metal ions (57).
Examination of ribozyme-catalyzed reactions in the presence
of various metal ions in combination should clarify the roles
of metal ions in catalysis.
ACKNOWLEDGEMENTS
The authors thank Dr Laura Nelson for helpful comments.
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Nucleic Acids Research, 2004, Vol. 32, No. 14
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